JPH02261370A - Cell-stimulating electric device - Google Patents

Abstract

PURPOSE:To prevent the precipitation of cells in a cell fusion chamber and cell-introducing tubes disposed near the inlet of the chamber by enabling the continuous rotation of both the chambers and the tubes. CONSTITUTION:A spacer 13 is nipped with a pair of electrode plates 12a and 12b, and electrode terminals 9a and 9b are pressed and brought into contact with the conducting films of the electrode plates. A cell-stimulating electric chamber can be rotated around rotation shafts 3a and 3b and a liquid inlet 12'b and a liquid outlet 12'a are connected with a liquid charging section 1b and a liquid discharging section 1a through tubes 11b and 11a to join to a liquid charging pipe 1'b and a liquid discharging pipe 1'a. A cyclic electrode holder 5 is disposed on the rotation shaft 3a and a pair of concentric cyclic electrodes are connected with terminals through conductor bars and further with the terminals 9a and 9b through lead lines. The terminals are pressingly brought into contact with a pair of external fixed electrodes with a spring.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a cell electrical stimulation device in which a cell electrical stimulation chamber is capable of continuous rotation, and this device uses a large amount of plant cell suspension. It is particularly useful for efficiently performing cell fusion by electrical stimulation.

[Conventional technology]

Cell fusion methods include chemical fusion methods and electrical fusion methods, but the former has drawbacks such as the polyethylene glycol (PEG) used for fusion being cytotoxic and low fusion efficiency. . On the other hand, in the latter case, there is no toxicity to cells and fusion can be achieved with high efficiency.

Normally, in the case of cell fusion using electrical stimulation, two types of cells (protoplasts) are first suspended in the space between a pair of parallel electrodes, and the cells are brought into contact by applying an alternating current voltage between the electrodes. This utilizes the phenomenon of dielectrophoresis, where cells are attracted to the direction of higher current density.
The resulting beads of cells are called pearl chains. Then, in this state, a DC pulse potential is applied to fuse the cells.

Conventionally, a cell fusion chamber in which a space having a liquid inlet and a liquid outlet is formed between a pair of parallel plate electrodes has been used as a means for continuously performing cell fusion using such electrical stimulation. However, plant protoplasts (cells) are prone to sedimentation, which occurs in a stationary camber or in an introduction tube, which tends to form pearl chains of 3 or more cells, and thus fusion of 3 or more cells. There was a drawback that multi-fusion was likely to occur. Furthermore, in conventional continuous cell fusion chambers, after one cell fusion operation is completed, the cell suspension in the chamber is taken out and at the same time a new cell suspension is introduced into the chamber for the next cell fusion operation. Since this is a semi-continuous method, there was a problem that during the cell fusion operation, cells in the cell suspension for the next cell fusion would settle in the introduction tube.

(Problem B to be Solved by the Invention) Therefore, the present invention makes it possible to prevent the precipitation of cells within the chamber during cell fusion by making the conventional cell fusion chamber capable of continuous rotation, and to prevent the precipitation of cells within the chamber during cell fusion. The above problems are solved by providing a cell fusion device using electrical stimulation that can prevent cells from settling in the tube during the waiting time by rotating the cell introduction tube near the chamber entrance simultaneously with the rotation. .

[Means to solve the problem]

The above problem is solved in a cell electrical stimulation device having a cell electrical stimulation chamber in which a space having a liquid inlet and a liquid outlet is formed between a pair of parallel plate electrodes. It is rotatable by a vertical rotation shaft, and each of the liquid inlet and liquid outlet is connected to a liquid inlet and a liquid discharge part provided at the outer end of the rotation shaft through a liquid flow path, and the liquid The injection section and the liquid discharge section are connected to a non-rotating liquid injection tube and a liquid discharge tube, each of the parallel plate electrodes is connected to a pair of annular electrodes that rotate with the rotation axis, and each of the parallel plate electrodes is connected to a pair of annular electrodes that rotate with the rotation axis. The solution is to provide a cell electrical stimulation device characterized in that the electrodes are in contact with a pair of external fixed electrodes.

[Operation and effect of the invention]

When using the above-described device, the parallel plate electrodes are typically placed parallel to gravity and rotated. Therefore, the cells do not settle and accumulate on the electrode surface, and the cells do not concentrate on a portion of the electrode surface, so that a suspension with a uniform concentration is maintained. Therefore, multi-fusion is suppressed and fusion between two cells can be efficiently performed. In addition, since the liquid flow path between the liquid inlet of the cell electrical stimulation chamber and the liquid injection part provided at the outer end of the rotation shaft rotates with the rotation of the chamber, cells waiting to be processed are not precipitated in the flow path. is prevented.

〔Example〕

Next, the apparatus of the present invention will be explained with reference to the drawings.

The cell electrical stimulation chamber used in the device of the present invention may have a similar structure to a conventional electrical stimulation chamber, and its structure is not a feature of the present invention; an example thereof is shown in FIG. In this figure, a spacer 13 having a hollow 13'' is sandwiched between a pair of electrode plates 12a and 12b to form a space therebetween for accommodating a cell suspension.The spacer 13 has small holes at opposing positions. It has holes 13'a and 13'b, and these small holes are hollow 1
3''.Each electrode plate has an electrode made of a conductive film attached to one surface of an insulator plate, and the conductive film of the pair of electrode plates is located inside the space. Each insulator plate has a liquid inlet 12'b and a liquid outlet 12'a, which correspond to the small holes 13'b and 1 in the spacer, respectively.
It is in contact with 3'a. If the spacer and each electrode plate are square, for example, their corners are cut off, and in this case the electrode plates 12a and 12b have their corners cut off at different positions. Further, the spacer is cut out at two corners corresponding to the cut corners of the electrode plate.

Therefore, when a spacer and two electrode plates are stacked on top of each other, the conductive film of each electrode plate is exposed to the outside at one corner. Then, the electrode terminal 9a comes into contact with the conductive film of the electrode plate 12b, and the electrode terminal 9b comes into contact with the conductive film of the electrode plate 12a. In this way, a cell electrical stimulation chamber is constructed in which a cell fusion space having a liquid inlet and a liquid outlet is formed between a pair of parallel plate electrodes.

Although an example of a cell electrical stimulation chamber has been described above, the chamber of the present invention is not limited to this. For example, the outer shape of the spacer and electrode plate need not be a square with cut corners, but may be rectangular or circular. , and various other shapes. Further, the hollow space of the spacer does not necessarily have to be circular, and may have any shape such as a square or a polygon. Further, the voltage supply system to the liquid inlet/outlet and the parallel plate electrodes may be constructed using other structures.

Said cell electrical stimulation chamber is made rotatable by rotation axes 3a and 3b perpendicular to its parallel plate electrodes.

According to one embodiment, the rotating shafts 3a and 3b have chamber holders 6a and 6b at their inner ends, and the chamber is held between the pair of chamber holders,
For example, 4 poles)? a to 7d and fasteners 15a to 15
The zero rotating shafts 3a and 3b, which are tightened by d, are passed through bearings 2a and 2b, for example, and these bearings are fixed to the base as shown in FIGS. 3 and 4.
The rotating shaft and the chamber holder are preferably formed integrally. The chamber holder 6a has protrusions 8a-8d, whereby the cell electrical stimulation chamber is attached to the holder 6a.
placed in place on the top. Note that a flexible packing 6'a can be inserted between the chamber holder 6a and the monopolar plate 12a, and in addition to having a hole in the center, this packing has a cutout corresponding to the groove 14a on the holder 6a. has. A similar seal (not shown) can be provided between chamber holder 6b and electrode 12b.

As shown in FIGS. 1 and 2, the liquid population 12'b and the liquid outlet 12'a are connected to a liquid inlet provided at the outer end of the rotating shaft by means of a liquid flow path consisting of, for example, flexible tubes llb and lla. 1b and the liquid discharge part 1a.

The liquid injection part 1b and the liquid discharge part 1a protrude outward from the end of the rotating shaft, and are connected to the liquid injection pipe 1'b and the liquid discharge pipe 1'a, respectively. This connection is made between the rotating liquid injection part 1b and liquid discharge part 1a and the non-rotating liquid injection pipe 1'b.
and the liquid discharge pipe 1'a may be watertightly connected, and various such connection means are known, and any one of these known means can be selected and used. 0 For example, it is sufficient that the liquid injection part 1b and the liquid injection pipe 1'b are in rotational sliding contact in a watertight manner, and the same applies to the liquid discharge system.

In use, liquid injection pipe 1'b and liquid discharge pipe 1'a
includes flexible tubes 1″b and l″ for the passage of liquid.
According to a preferred embodiment of the present invention, a flexible tube Il connects the liquid inlet/outlet of the cell electrical stimulation chamber and the liquid inlet/outlet at the outer end of the rotation shaft.
As shown in FIGS. 1 and 2, a and Ilb are passed through the rotating shaft through the hollow L (10a and 10b). For this purpose, a groove 14a is provided on the inner surface of the chamber holder 6a to accommodate the tube lla, for example. Similarly, chamber holder 6 is used to accommodate tube Ilb.
A groove is provided on the inner surface of b (not shown).

In another embodiment, the tubes lla and llb are once taken out of the rotating shaft through holes penetrating the chamber holders 6a and 6b, and then the liquid discharge part 1a and the liquid It can also be connected to the injection part 1b.

An annular electrode holder 5 is provided on the rotating shaft 3a, and this holder 5 is preferably constructed integrally with the rotating shaft 3a.

This annular electrode holder 5 has a pair of concentric electrodes 4a and 4b (see FIG. 4). These electrodes 4a and 4b are conductor rods 4'a that pass through the electrode holder.
and 4'b to the terminals 16a and 16b. The terminal 9a in contact with the conductive film of the electric scraper 12b is a lead &
'i9'a is connected to the terminal 16a, while the terminal 9b in contact with the conductive film of the electrode plate 12a is connected to the terminal 16b by the lead wire 9'b. The lead wire 9'b passes through a hole 9''b penetrating the chamber holder 6b and a hole 16'b penetrating the chamber holder 6a. Alternatively, one such strip-shaped electrode may be provided on the periphery of each of the rotating shafts 3a and 3b.

The rotating structure configured as described above is attached to a pedestal at its bearings 2a and 2b as shown in FIGS. 3 and 4. This pedestal is 20a and 20
It is composed of a retaining plate consisting of 1.b and fixing rods 21a and 21b that fix these plates at regular intervals. This fixation is accomplished by conventional means. The spacing between the holding plates is made equal to the spacing between the bearings 2a and 2b of the rotating shaft. These retaining plates have, near their centers, notches 20'a and 20'b (not shown) of the same depth as the diameter of the bearings 2a and 2b, into which the bearings are fitted. These bearings are attached to the holding plates 22a and 2
2b and fasteners 22'a and 22'b (not shown) to the retaining plate. Holding plate 20a
and 20b have incisions 20'a and 2 near their ends.
It has cuts 26a1 and 26b (not shown) in the direction opposite to 0'b, which are fitted into the retaining post 25 of the drive part.

Fixed electrodes 18a and 18b are attached to the holding plate 20a, and these include contact electrodes 17a and 17b, which are insulated from the holding plate 20a by an insulator, and a terminal 19a.
and 19b are inserted. Terminal 19a and contact electrode 1
7a and between the terminal 19b and the contact electrode 17b, the contact electrodes 17a and 17b slide into contact with the annular electrode attached to the electrode holder 5.

The drive unit has drive wheels 23a and 23b provided on the drive control unit 27, as shown in FIG. One edge is in contact and rotation of the drive wheel rotates said rotating structure.

In the above explanation, it is assumed that the liquid is injected from the upper part and the liquid is discharged from the lower part with respect to FIGS. Since the device is used lying down, it is also included in the mode of use of the present invention even if the directions of liquid injection and discharge are reversed.

[Brief explanation of drawings]

FIG. 1 is an exploded perspective view of the rotating structure of the device of the present invention. FIG. 2 schematically shows a cross section of the rotating shaft and holder of the device of the present invention. FIG. 3 is a plan view showing a state in which the rotating structure portion of the device of the present invention is set on a pedestal. FIG. 4 is a front view showing the assembled state of the device of the present invention. In the figure, 1a is a liquid discharge (injection) part, 1'a is a liquid discharge (injection) tube, 3a and 3b are rotating shafts, 4a and 4b are ring electrodes, 5 is a ring electrode holder, 6a and 6b are cell electrical stimulation Chamber holder 11! and llb are liquid conduits, 12a and 12b are electrode plates, and 13 is a spacer.

Claims (1)

[Claims] 1. In a cell electrical stimulation device having a cell electrical stimulation chamber in which a space having a liquid inlet and a liquid outlet is formed between a pair of parallel plate electrodes, the cell electrical stimulation chamber is rotated perpendicularly to the parallel plate electrodes. The liquid inlet and the liquid outlet are each connected to a liquid inlet and a liquid outlet provided at an outer end of the rotating shaft through a liquid flow path, and the liquid inlet and the liquid outlet are connected to each other through a liquid flow path. The liquid discharge part is connected to a non-rotating liquid injection tube and a liquid discharge tube, each of the parallel electrodes is connected to a pair of annular electrodes that rotate with the rotation axis, and the pair of annular electrodes are connected to a pair of annular electrodes that rotate with the rotation axis. A cell electrical stimulation device, characterized in that it is in contact with an externally fixed electrode.